This project aims to advance understanding of the mode of action of the clinically vital but also highly toxic antifungal drug amphotericin B (AmB). Alternative to the classic ion channel model, preliminary studies in this proposal show that AmB forms large extramembranous aggregates that extract sterols from lipid bilayers and thereby kill cells. This novel sterol sponge model illuminates a new and more actionable roadmap to an improved therapeutic index, i.e., maximize the relative binding affinity of the AmB sterol sponge for the sterol found in fungi (ergosterol) vs. humans (cholesterol). Aim 1 is to determine the structure of the AmB sterol sponge, assembled in the presence of physiologically relevant lipid bilayers. Aim 2 is to determine the structures of the complexes of the AmB sponge with ergosterol and cholesterol. Aim 3 is to determine the structure of the sterol sponge and corresponding ergosterol complex derived from a new derivative of AmB, AmBMU, which was recently discovered and shown to bind ergosterol but not cholesterol, to be non-toxic to human cells, and to retain potent antifungal activity in vitro and in mice. Collectively, these studies wll provide a high-resolution picture of the atomistic interactions that underlie the biological activities of AmB and thus powerfully enable the rational development of less toxic derivatives of this clinically vital natural product. These studies will also further illuminate the fundamental features of how clinically validated resistance-refractory antimicrobial action can be achieved and lay the foundation for the frontier pursuit of other biologically relevant small molecule-small molecule interactions. Relevance to Human Health. Amphotericin B is the powerful but unfortunately highly toxic gold standard therapy for treatment of systemic fungal infections, and this drug has uniquely evaded the emergence of microbial resistance despite more than half a century of widespread clinical utilization. Better understanding how AmB exerts its biological activities is thus critical for guiding the rational development of derivatives with an improved therapeutic index as well as other resistance-refractory antimicrobial agents.